MicroRNAs (miRNAs) are small RNAs (~22 nucleotides) that regulate gene expression by inhibiting protein translation or by degrading mRNA transcripts. The miRNA gene regulatory pathway is highly evolutionarily conserved and is emerging as a major player in immune cell development and function, by controlling a diverse array of biological processes such as cell signaling, proliferation and apoptosis. We have recently shown that the miRNA repertoire of CD8 T cells is dynamic and rapidly changes after infection as naive cells get activated and differentiate into effector cells and then memory cells. In particular, we found that the miR- 17~92 cluster is highly upregulated in rapidly expanding effector cells, with relatively lower expression in memory-fated (memory precursor effector cells, MPECs) than short-lived effector cell (SLECs) subsets. Consistent with our hypothesis that miR-17~92 may be regulating effector and memory fates, deletion or constitutive expression of miR-17~92 revealed a critical CD8 T cell-intrinsic role of miR-17~92 in driving terminal effector differentiation by promoting proliferation. These studies lay a strong framework for investigating the molecular mechanisms by which miR-17~92 regulates effector and memory fates. Using CD8 T cell-specific gain or loss of expression of miR-17~92, we have compelling preliminary evidence supporting our hypothesis that miR-17~92 decreases the threshold of T cell activation by antigen, thus promoting terminal effector differentiation through excessive stimulation and proliferation: (i) Loss of miR- 17~92 impacts CTL expansion and survival, in a manner that is dependent on systemic levels of antigen; (ii) Inhibitors of T cell signaling (the tumor suppressor protein, PTEN, and inhibitory receptor PD-1) are decreased upon overexpression of miR-17~92; (iii) T cell activation by antigen is enhanced upon overexpression of miR- 17~92. Under the R03 funding mechanism, the goal of this small self-contained research project is to conduct incisive experiments that will definitively evaluate the role of miR-17~92 on T cell signaling threshold in antigen-specific CD8 T cells, and also identify key signaling pathways and proteins that are regulated by miR- 17~92. We will measure TCR sensitivity in our dual model of gain or loss of expression of miR-17~92 using suboptimal antigenic peptide dose as well as altered peptide ligands of varying affinity; we will quantify signal transduction cascades downstream of TCR stimulation upon overexpression or loss of miR-17~92; and we will conduct gene profiling of effector CD8 T cells lacking or overexpressing miR-17~92 during early stages of activation towards potentially identifying novel miR-17~92 targets involved in regulating strength of T cell stimulation, proliferation and terminal effector differentiation. Induction of potent memory CD8 T cells is an important goal of vaccination. Thus, greater understanding of molecular regulation of effector and memory lineage specification, as resulting from proposed studies, has direct relevance to manipulation of CD8 T cell immunity during tumor outgrowth, infection and vaccination.